Mouse cd4 beads

Spleens were extracted from adult C57BL6 wild type mice and mechanically dissociated as described in [26 (link), 27 (link)]. After red cell lysis in ammonium chloride potassium lysis buffer (Sigma-Aldrich), spleen cells were resuspended in complete T cell medium (RPMI medium supplemented with 1% Pen/Strep solution, 200 mM l-Glutamine, 100 mM Sodium Pyruvate and 5% FCS). CD4 + T lymphocytes were purified from spleens of 2D2 mice by magnetic sorting with mouse CD4 + beads according to manufacturer’s instructions (Miltenyi Biotec). For differentiation in distinct Th subsets, CD4 + T cells were maintained in complete T cell medium in 96-well round-bottom plates at 200.000 cells/well. Cells were stimulated with anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) antibodies (BD Biosciences) and left grown for 7 days in the presence of either IL-12 (10 ng/ml) and anti–IL-4 (5 µg/ml; Th1 differentiating conditions), IL-4 (10 ng/ml) and anti-IFN-γ (10 ng/ml; Th2 differentiating conditions) or TGF-β (3 ng/ml) and IL-6 (30 ng/ml; Th17 differentiating conditions).